In respect to clinical investigations, the proportion of DNA-synthesizing cells (S-phase cells) in tumors has been used as a measure of their proliferation rate. The DNA-synthesizing cells have earlier been determined by means of radioactive labelled thymidine (autoradiography). Recently, even incorporation of halogene-analogs of thymidine (BrdU) and antibodies against these have been used and, to a large extent, quantitative flowcytometric DNA measurements. The disadvantage with the use of isotopic labelled thymidine and BrdU is the fact that only living cells can be measured. Therefore, these methods have only been used on selected patients and in a limited number of studies (1). Flow-cytometry, a technique working with fixed non-living cells, but also with stored cell material, has high capacity, but is unable to distinguish between proliferating and non-proliferating S-phase cells. Determination of S-phase cells is even more complicated in tumors containing both normal and malignant cells which do not deviate in their DNA-content from benign cells (2). Other methods for determination of markers specifically related to proliferating cells are known. For example, Ki-67 and PCNA (3). Ki-67 is a monoclonal antibody against a substance in the cell nucleus. The exact nature of Ki-67 is still unknown. Ki-67 is expressed in all cell cycle stages except in G.sub.o and early G.sub.1, with a maximum in G.sub.2 and mitosis. One type of antibody available on the market can only be used in fresh tissues, one newer type also in formalin fixed, paraffine embedded tissues.
PCNA is a nuclear protein (36 kD) identical with that of the subunit of DNA-polymerase delta. Depending on the cell proliferation rate, PCNA is expressed in all cell cycle stages. PCNA is not as sensitive for various fixation techniques as Ki-67 (3).
A problem with Ki-67 and PCNA is that these substances are expressed in all cell cycle stages and thus only to some extent reflected the proportion of S-phase cells.
Thymidine kinases appear in two different forms, a cytoplasmic (TK1) (4) and a mitochondrial (TK2) (5) form, with different genetic origin. TK1, which can be subdivided into different variants (6), is closely related to cell proliferation and starts to be synthesized at the border of G.sub.1 /S (7). TK1 is degraded in connection with mitosis (8). Thus, TK1 is a specific marker for S and G.sub.2 cells.
There have been several attempts to make TK1 specific antibodies with the intact protein and various recombinant forms. However, due to the fact that the primary structure of TK1 is highly conserved between different species there are very few antibodies reported and the antibodies produced show extensive cross reactivity to other proteins (8, 9).
EP A1 255 431 describes a foetal form of thymidine kinase (TK-F) which is said to have a superior enzymatic activity compared to previously known TK-F. Moreover, the applicant describes a purification procedure and use of TK-F to produce antibodies. These antibodies are said to have a surprising effect on patients with hormone dependent cancer, especially prostate and breast cancer, respectively. Diagnostic use of said antibodies is solely mentioned and the application describes nothing further in this respect.
EP A2 137 492 describes a process for early diagnosing of malignant tumors with antibodies directed against different blood forms of nucleoside-nucleotide phosphotransferases. Here not only thymidine kinase is determined but also several other phosphotransferases which means that the process is not specific for thymidine kinase.
The present invention makes it possible to estimate the proportion of proliferating S and G.sub.2 cells by determining the cellular concentration of the cytosolic thymidine kinase. This determination is enabled by using antibodies against a defined part of TK 1, namely a 15 amino acid residues peptide. The antibodies according to the invention do not produce any cross reactivity with other proteins.
The peptide sequence according to the invention represents a unique structure at the C-terminal end of the protein which is most likely part of an external bend region of the protein. It is also known that C-terminal sequences are involved in cell cycle specific degradation of TK1 (8). Therefore, this amino acid sequence is most likely specifically immunogenic and the corresponding epitope of TK1 is exposed and present only in intact TK1.
Measurements of TK1 activity in serum of patients with malignancies have been extensively used as a marker of disease (10). However the TK1 activity determinations by radioactive substrate assay is relatively complex. Therefore, there is a need for simplified quantitative assays for TK1 that could be used in cell extracts, cells, tissues and body fluids.
This need is fulfilled according to the invention by antibodies that are unique and can serve as specific markers for proliferating cells, since the antigen is expressed only in S and G.sub.2 cells. These antibodies can be used to determine the degree of proliferation in any tumor and the antibodies have been used with both fresh and stored paraffine embedded cell material. The antibodies can react with TK1 also after various types of fixation.